Project Summary: Future tactics for restoration of hearing or balance disorders in humans may target developmental signaling to reactivate progenitor character in native cells or utilize directed differentiation of pluripotent stem cells to produce transplantable sensory cells or tissues. These approaches will involve the generation of cellular states with characteristics similar to the prosensory progenitors that give rise to auditory and vestibular sensory organs during embryogenesis. A barrier faced here is our lack of information regarding the transcriptional programs that specify early otic progenitors toward vestibular vs. auditory and prosensory vs. non-sensory fates. Goals for this project include discovery of genetic mechanisms of auditory and vestibular prosensory specification and comparative systems-level evaluation of transcriptional programs active in in vitro generated otic sensory cells. These efforts will support the candidate's long-term objective of producing auditory or vestibular organ-specific progenitors in vitro and eventual bioengineering of transplantable organs. Specific aims for achieving the stated goals include: 1) transcriptomic and epigenomic profiling of auditory and vestibular sensory cells and prosensory progenitors, 2) functional testing of regulatory mechanisms of auditory and vestibular prosensory genes, and 3) transcriptomic and epigenomic profiling of in vitro generated otic sensory cells. Innovations in research design include: novel Fbxo2 knock-in reporter mice and ES cell lines for specific otic sensory lineage delineation in live cells, the use of a combination of recombinase-editable transgenic mouse models and explant organ culture for regulatory element testing, and the application of recently developed advances in RNA-seq and ATAC-seq for transcriptional and epigenomic analysis at the single cell level. Accomplishing the above specific aims will uncover mechanisms behind key fate decisions that lead to formation of auditory and vestibular organs and provide a direct comparison to in vitro generated otic organoid cells. The candidate is well suited to the project, with expertise in inner ear development and gene regulation and a strong record of training and past research accomplishments. The scientific environment for the mentored phase of this project, the Department of Otolaryngology at Stanford University School of Medicine, provides strong resources and collaboration opportunities to contribute to the projects success. This ECR R21 funding will support the candidate's transition to independence and generate papers and preliminary data for a first RO1. Future work in the candidate's lab will build upon this topic with parallel studies in mouse and ES cell models to investigate lineage specification, fate determination, and sensory organ bioengineering. This proposal represents a first step in what the candidate is confident will be a long journey of substantial contribution to science within the mission of the NIDCD.